Wheat whole genome liquid phase chip and application thereof

Abstract

The invention discloses a wheat whole genome liquid phase chip and application thereof. The liquid-phase chip has 175 foreground detection sites and 4, 350 background detection sites, the foreground detection sites comprise genes / QTL related to characters such as gibberellic disease resistance, rust disease resistance, powdery mildew resistance, yield, plant height, quality, grain development and pre-harvest sprouting resistance, and genotypes of multiple different functional sites can be subjected to molecular detection at the same time; the background detection site is suitable for genetic background screening and evaluation of wheat germplasm resources, construction of linkage genetic maps, QTL positioning and other researches.

Description

Technical Field

[0001] The invention belongs to the technical field of whole genome gene chips, and specifically relates to a wheat whole genome liquid phase chip developed based on targeted capture sequencing technology, and the application of the liquid phase chip in wheat. Background Art

[0002] Wheat is an important food crop in the world and the staple food for 30% of the world's population. It provides calories and protein consumed by 2.5 billion people worldwide. Currently, due to the limitations of breeding technology, traditional breeding takes a long time, and it takes about 8-10 years to breed a variety. The efficiency is low, and phenotypic identification is time-consuming and labor-intensive. Molecular breeding helps to break through the limitations of conventional breeding. Using markers that are closely linked to the target gene, selection can be performed in early generations, thereby shortening the breeding cycle and improving breeding efficiency. It is crucial to improve wheat breeding problems and accelerate molecular-assisted breeding by combining molecular breeding and chip technology.

[0003] Wheat has a complex and huge genome, and also has a high proportion of repetitive sequences or transposons, which are highly polymorphic in different materials. Single nucleotide polymorphisms (SNPs) refer to sequence polymorphisms caused by the conversion, transversion, deletion or insertion of a single nucleotide in the genome. This polymorphism is widely present in the whole genome of organisms, including gene coding regions and non-coding regions, with a high SNP density; strong polymorphism and high frequency, combined with chip and other detection technologies, is conducive to the development of automated, high-throughput technology screening and detection; and low mutation and high stability. Especially for the wheat genome, it has the advantages of ultra-high throughput detection and easy integration of genotype data, and is an ideal genotyping marker.

[0004] Recently, genotyping technology based on targeted capture sequencing (GBTS) has been gradually applied to animals and plants. It selects specific target sites from genomic DNA for sequencing and genotype detection. It consists of two technical systems: the GenoPlexs system based on multiplex PCR and the GenoBaits system based on liquid phase probe hybridization. Its working principle is to design target probes that are complementary to the target sequence bases, thereby achieving fixed-point capture of specific regional sequences, and combined with high-throughput sequencing, to achieve target fragment site detection and variation analysis. Compared with solid-phase chips, GBTS marker development technology is simple, and the development, testing, and verification costs are much lower than traditional solid-state chips; the density of SNP chips can be flexibly adjusted, and marker sites can be upgraded and added at any time, and it is not limited by the amount of samples tested; this technology is applicable to all biological species and can be widely used in resource evaluation, gene positioning and cloning, genetic map construction, molecular marker-assisted selection, variety protection and other fields. At present, single nucleotide liquid phase chips based on GBTS technology have been developed and used in plants such as barley, cotton, corn, rice, and soybeans. Most of the current wheat chips are solid-state chips, and are designed for background screening. There is currently a lack of an accurate, efficient, and low-cost wheat chip that can be used for both foreground selection and background selection. Summary of the invention

[0005] The object of the present invention is to develop a 5K wheat whole genome liquid phase chip using targeted sequencing genotyping (GBTS) technology. The chip includes sites for prospect detection of known genes / QTLs and sites for genetic background detection in the wheat genome. The prospect detection sites include genes of more than 100 important breeding targets such as disease resistance, yield, quality, plant height and anti-ear sprouting. The chip can be used for molecular assisted breeding of target traits, and can also be used for background recovery rate detection when constructing near-isogenic lines. In addition, we can use genotype data for QTL positioning, genetic diversity analysis and background selection. The greatest significance of this chip is to convert a large number of molecular markers into breeding chips, while providing background detection and prospect detection for molecular breeding. Breeders can use the genotyping data of the chip and the introduction of genomic selection to achieve multiple gene accumulation and transfer, which helps to promote the molecular breeding technology to select excellent varieties.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] In a first aspect, the present invention provides a wheat whole genome liquid phase chip, which includes 5K wheat SNP site probes, wherein the 5K wheat SNP site probes include 175 foreground detection site probes and 4,350 background detection site probes, wherein the foreground detection sites include genes / QTLs related to traits such as resistance to ergot, rust, powdery mildew, yield, plant height, quality, grain development and anti-ear sprouting, and can realize molecular detection of genotypes of multiple different functional sites at the same time; the background detection sites are screened based on the principle of high polymorphism rate in materials and uniform distribution between chromosomes, using genome resequencing data of 68 materials with physical location information of IWGSC Ref Seq v2.1 as a reference; the number of background selection markers finally obtained can ensure a probability of more than 99% to obtain 200 polymorphic markers between any two materials, and the background detection is suitable for genetic background screening and evaluation of wheat germplasm resources, construction of linkage genetic maps, QTL positioning and other studies.

[0008] In a specific embodiment, the wheat reference genome is the whole genome sequence of Chinese Spring, version number IWGSC Ref Seq v2.1. The 175 foreground detection sites are shown in Table 1, and the 4,350 background detection sites are shown in Table 2.

[0009] Among them, 1A~7A, 1B~7B, 1D~7D indicate the chromosomes where the sites are located, and the subsequent values ​​are the corresponding sites of the wheat liquid phase probe on each chromosome on the wheat reference genome. For example, Chr1A indicates chromosome 1A of wheat, and 371520694 indicates that it is site 371520694 of chromosome 1A of wheat.

[0010] In a specific embodiment, each site of the liquid phase chip contains at least 2 liquid phase probes.

[0011] In a specific embodiment, the liquid phase chip further comprises a hybridization capture reagent.

[0012] The probe design process of wheat whole genome liquid phase chip is as follows:

[0013] (1) The probe length was 110 bp, the GC content of the probe was between 30% and 80%, the number of homology regions was ≤ 5, and the selected region did not contain SSR and GAP regions as much as possible;

[0014] (2) Designing two nucleotide sequences that overlap by 60% to 70% and cover the oligonucleotide probe sites based on the oligonucleotide probe sites obtained by screening.

[0015] In the second aspect, the present invention also protects the use of the wheat whole genome liquid phase chip mentioned above in wheat whole genome selection breeding.

[0016] In a third aspect, the present invention also protects the use of the wheat whole genome liquid phase chip mentioned above in the preparation of wheat whole genome selection breeding products.

[0017] In a fourth aspect, the present invention also protects the use of the wheat whole genome liquid phase chip mentioned above in wheat genetic diversity analysis.

[0018] In a fifth aspect, the present invention also protects the use of the wheat whole genome liquid phase chip mentioned above in wheat variety identification.

[0019] In a sixth aspect, the present invention also protects the use of the wheat whole genome liquid phase chip mentioned above in wheat kinship identification.

[0020] In a seventh aspect, the present invention also protects the use of the wheat whole genome liquid phase chip mentioned above in wheat whole genome association analysis.

[0021] The beneficial effects of the wheat whole genome liquid phase chip of the present invention are:

[0022] (1) The present invention utilizes wheat genome and resequencing data to identify wheat-specific regions at the whole genome level through bioinformatics methods, designs liquid phase chip oligonucleotide probes for gene regions or adjacent regions, achieves large-scale target fragment capture, and focuses on covering disease resistance and high-quality gene regions with significant application value;

[0023] (2) This liquid phase chip is developed based on targeted capture technology. The working principle is to design probes and test sample DNA for targeted capture and sequencing through base complementary pairing, thereby achieving the purpose of gene detection and typing in the target area;

[0024] (3) Compared with solid-phase chips, this liquid-phase chip has higher detection accuracy, more effective information, higher flexibility and diversity, is applicable to a wider range of sequencing platforms, has a shorter test cycle, and significantly reduces the detection cost, providing important technical support for wheat genetic improvement and helping to promote the use of molecular breeding technology to select superior wheat varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The distribution map of 175 prospect detection sites of the SNP markers of the present invention on chromosomes;

[0026] Figure 2 is the distribution map of the SNP markers of the present invention on chromosomes;

[0027] Figure 3 This is a relationship diagram of different wheat parent breeding materials in Example 3 of the present invention. DETAILED DESCRIPTION

[0028] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1

[0030] A wheat whole genome 5K liquid phase chip based on targeted sequencing genotyping (GBTS) technology, including 175 foreground detection site probes and 4,350 background detection site probes, each SNP site contains two different base variation sites for detecting allele changes at the site, and the physical position of the SNP site is determined based on the sequence alignment of the wheat reference genome IWGSC Ref Seq v2.1. The wheat reference genome IWGSC Ref Seq v2.1 sequence can be obtained from the website: https: / / www.wheatgenome.org / ; The variation information of the SNP site is expressed in the form of chromosome_physical position reference genotype / variant allele type; among them, the variation information of 175 SNP prospect detection sites is shown in Table 1, and the variation information of 4,350 SNP background detection sites is shown in Table 2.

[0031] Table 1 Variation information of 175 SNP prospect detection sites

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[0035] Table 2 4,350 SNP background detection sites

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[0052] The method for obtaining a combination of 175 SNP loci related to wheat breeding traits is as follows: (1) Develop markers based on the specific sequences of cloned ergot resistance genes and genes controlling other wheat traits; (2) Screen multiple SNPs with high specificity or polymorphism within the QTL positioning interval and convert them into chip markers to achieve haplotype analysis of chromosome segments; (3) Convert KASP markers and STARP markers verified in other studies into chip markers with gene / QTL identification functions.

[0053] Effect verification: Analyze the collected SNP loci and important breeding traits, and screen out SNP loci with excellent trait performance. Among them, the important breeding traits include resistance to ergot, stripe rust, powdery mildew, leaf rust, yield, plant height, ear development, stress resistance, vernalization and ear germination.

[0054] The preparation method of the wheat whole genome liquid phase chip is as follows:

[0055] (1) The probe length was 110 bp, the GC content of the probe was between 30% and 80%, the number of homology regions was ≤ 5, and the selected region did not contain SSR and GAP regions as much as possible;

[0056] (2) Based on the probe sites obtained through screening, two nucleotide sequences with 60% to 70% overlap and covering the probe sites are designed.

[0057] The distribution of 175 SNP loci on chromosomes was analyzed. Figure 1 ; The distribution of 4350 background detection sites on chromosomes, the results are shown in Figure 2 .

[0058] Example 2

[0059] The above 175 prospect detection sites were used to detect functional genes in breeding materials, and more than 50 wheat varieties with known genotypes were used for comparison to verify the genotypes of these genes. The results showed that the chip of the present invention can be used for molecular marker-assisted selection of target breeding. The specific steps include:

[0060] Step 1. Extraction and quality inspection of sample DNA: Plant the verified materials in plug trays, extract plant genomic DNA using the SDS method from two-leaf, one-heart young leaves, use 1% agarose gel electrophoresis to detect the quality of sample DNA, use Qubit Flex to accurately determine the genomic DNA concentration, and adjust the DNA concentration to 100 ng / ul.

[0061] Step 2, library construction: After DNA quality inspection, use Twist Library Preparation EF Kit 2.0 (104207) library construction kit, take 100ng gDNA and use enzyme digestion to fragment, fill the ends of the fragmented DNA, and phosphorylate the 5' end. At the same time, the fragmented product is repaired and dA tailed, and connected to the universal sequencing adapter. Use magnetic beads to purify and sort the fragments, and retain the adapter connection product with an insert of 200-300bp. Add sequencing primers (with different barcodes to distinguish different samples) to the connection product and use a high-fidelity PCR reaction system for PCR amplification. The amplified product after magnetic bead purification is the final probe capture pre-library, which can be used for probe capture experiments.

[0062] Step 3, probe capture: After the pre-library quality inspection is completed, the library samples to be captured are mixed in equimolar amounts to form a DNA library pool. After the library pool is dried, the acquisition probe and capture reagent are added to denature it and reacted at 60°C for 2-16 hours. The hybridization capture product is enriched and washed with streptavidin magnetic beads, then PCR amplified and purified using magnetic beads to obtain the final capture library.

[0063] Step 4, sequencing: Qubit was used to quantify the captured library, and the library fragment size was detected using Agilent 4200. After passing the quality inspection, DNBSEQ-T7 was used for sequencing.

[0064] Step 5, alignment: Use fastp software (version 0.19.5) to process the original sequencing reads and remove low-quality reads and adapter sequences. Use BWA software to align it with the wheat Chinese Spring genome, and use bedtools software to calculate the sequence coverage of each probe site after obtaining the alignment file of each sample; use Samtools (version 1.9) to sort the BAM files of the alignment results, remove PCR duplications, and calculate alignment statistics. Use GATK (version 4.1.3.0) to generate the original population VCF file.

[0065] Step 6: Conduct molecular breeding of wheat using the materials containing excellent genes detected.

[0066] Step 7: Based on the relationship between genotype and advantageous breeding traits, the materials obtained through molecular breeding are used to determine the advantageous breeding traits of the offspring samples, and the parent materials with better comprehensive traits of the offspring samples are determined, thereby realizing molecular marker-assisted selection breeding.

[0067] Example 3

[0068] Using the 5K liquid phase chip of the present invention, 10 different parent wheat varieties were analyzed for kinship, and the specific steps included:

[0069] Step 1, select 10 different parent materials for DNA extraction: plant 10 materials in a plug tray, take two leaves and one heart of young leaves to extract plant genomic DNA by SDS method, use 1% agar gel electrophoresis to detect the quality of sample DNA, use Qubit Flex to accurately determine the genomic DNA concentration, and adjust the DNA concentration to 100ng / ul; Step 2, repeat the steps in Example 2; Step 3, repeat the steps in Example 2; Step 4, repeat the steps in Example 2; Step 5, repeat the steps in Example 2; Step 6, analyze different parent materials, and provide a reference for breeders to select materials and simply follow up the relationship. It is found that the pedigree relationship of common wheat varieties is correct, as shown in the following example. Figure 3 shown.

[0070] In summary, the wheat whole genome liquid phase chip of the present invention has the following advantages in terms of application.

[0071] (1) Using wheat genome and resequencing data, we identified wheat-specific regions at the whole genome level through bioinformatics methods. High-density single nucleotide polymorphism (SNP) chips facilitated genome-wide association and linkage analysis to detect the association between variant sites and phenotypes. We also located QTLs related to important agronomic traits such as disease resistance and yield, and constructed linkage genetic maps.

[0072] (2) This liquid phase chip is developed based on targeted capture technology. The working principle is to design probes and test sample DNA for targeted capture and sequencing through base complementary pairing, thereby achieving the purpose of gene detection and typing in the target area;

[0073] (3) Compared with solid-phase chips, the 5K wheat liquid-phase chip has high detection accuracy, more effective information, high flexibility and diversity, is applicable to a wider range of sequencing platforms, has a shorter test cycle, and significantly reduces the detection cost. In addition, the chip contains 175 foreground detection sites and 4350 background detection sites at the same time, which efficiently integrates and utilizes molecular marker information from different platforms, and mines the variation sites of target traits through genome-wide association and linkage analysis. At the same time, molecular marker-assisted selection can accelerate the screening and improvement of target traits, thereby shortening the breeding cycle, providing important technical support for wheat genetic improvement, and helping to promote the selection of excellent wheat varieties using molecular breeding technology.

[0074] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A wheat whole genome liquid phase chip, characterized in that: The wheat whole genome liquid phase chip includes 5K wheat SNP site probes, wherein the 5K wheat SNP site probes include 175 foreground detection site probes and 4,350 background detection site probes. The 175 foreground detection sites are as follows: The 4,350 background detection sites are as follows: The wheat reference genome is the whole genome sequence of Chinese Spring, version number IWGSC Ref Seq v2.

1.

2. The wheat whole genome liquid phase chip according to claim 1, characterized in that: Each site of the liquid phase chip contains at least two liquid phase probes.

3. The wheat whole genome liquid phase chip according to claim 1, characterized in that: The liquid phase chip also contains a hybridization capture reagent.

4. The method for preparing the wheat whole genome liquid phase chip according to any one of claims 1 to 3 is as follows: (1) The probe length was 110 bp, the GC content of the probe was between 30% and 80%, the number of homology regions was ≤ 5, and the selected region did not contain SSR and GAP regions as much as possible; (2) Based on the probe sites obtained through screening, two nucleotide sequences with 60% to 70% overlap and covering the probe sites are designed.

5. Use of the wheat whole genome liquid phase chip according to any one of claims 1 to 3 in wheat whole genome selection breeding.

6. Use of the wheat whole genome liquid phase chip according to any one of claims 1 to 3 in preparing products for wheat whole genome selection breeding.

7. Use of the wheat whole genome liquid phase chip according to any one of claims 1 to 3 in wheat genetic diversity analysis.

8. Use of the wheat whole genome liquid phase chip according to any one of claims 1 to 3 in wheat variety identification.

9. Use of the wheat whole genome liquid phase chip according to any one of claims 1 to 3 in wheat kinship identification.

10. Use of the wheat whole genome liquid phase chip according to any one of claims 1 to 3 in wheat whole genome association analysis.

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